The dispersal between Amazonia and Atlantic Forest during the Early Neogene revealed by the biogeography of the treefrog tribe Sphaenorhynchini (Anura, Hylidae)

Abstract The Amazonia and the Atlantic Forest, separated by the diagonal of open formations, are two ecoregions that comprise the most diverse tropical forests in the world. The Sphaenorhynchini tribe is among the few tribes of anurans that occur in both rainforests, and their historical biogeographic have never been proposed. In this study, we infer a dated phylogeny for the species of the Sphaenorhynchini and we reconstructed the biogeographic history describing the diversification chronology, and possible patterns of dispersion and vicariance, providing information about how orogeny, forest dynamics and allopatric speciation affected their evolution in South America. We provided a dated phylogeny and biogeography study for the Sphaenorhynchini tribe using mitochondrial and nuclear genes. We analyzed 41 samples to estimate the ancestral areas using biogeographical analysis based on the estimated divergence times and the current geographical ranges of the species of Sphaenorhynchini. We recovered three characteristic clades that we recognize as groups of species (S. lacteus, S. planicola, and S. platycephalus groups), with S. carneus and G. pauloalvini being the sister taxa of all other species from the tribe. We found that the diversification of the tribe lineages coincided with the main climatic and geological factors that shaped the Neotropical landscape during the Cenozoic. The most recent common ancestor of the Sphaenorhynchini species emerged in the North of the Atlantic Forest and migrated to the Amazonia in different dispersion events that occurred during the connections between these ecoregions. This is the first large‐scale study to include an almost complete calibrated phylogeny of Sphaenorhynchini, presenting important information about the evolution and diversification of the tribe. Overall, we suggest that biogeographic historical of Sphaenorhynchini have resulted from a combination of repeated range expansion and contraction cycles concurrent with climate fluctuations and dispersal events between the Atlantic Forest and Amazonia.

we infer a dated phylogeny for the species of the Sphaenorhynchini and we reconstructed the biogeographic history describing the diversification chronology, and possible patterns of dispersion and vicariance, providing information about how orogeny, forest dynamics and allopatric speciation affected their evolution in South America.
We provided a dated phylogeny and biogeography study for the Sphaenorhynchini tribe using mitochondrial and nuclear genes. We analyzed 41 samples to estimate the ancestral areas using biogeographical analysis based on the estimated divergence times and the current geographical ranges of the species of Sphaenorhynchini. We recovered three characteristic clades that we recognize as groups of species (S. lacteus, S. planicola, and S. platycephalus groups), with S. carneus and G. pauloalvini being the sister taxa of all other species from the tribe. We found that the diversification of the tribe lineages coincided with the main climatic and geological factors that shaped the Neotropical landscape during the Cenozoic. The most recent common ancestor of the Sphaenorhynchini species emerged in the North of the Atlantic Forest and migrated to the Amazonia in different dispersion events that occurred during the connections between these ecoregions. This is the first large-scale study to include an almost complete calibrated phylogeny of Sphaenorhynchini, presenting important information about the evolution and diversification of the tribe. Overall, we suggest that biogeographic historical of Sphaenorhynchini have resulted from a combination of repeated range expansion and contraction cycles concurrent with climate fluctuations and dispersal events between the Atlantic Forest and Amazonia.

| INTRODUC TI ON
The Neotropical region is the most diverse region on Earth, having from recent to old geological features, covering a wide range of geological and geomorphological formations Saadi, 1995) and the speciation time among organisms in this region has been widely debated (e.g., Batalha-Filho et al., 2012;de Sá et al., 2019;Fouquet, Loebmann, et al., 2012;Fouquet, Recoder, et al., 2012;Hoorn, Wesselingh, Hovikoski, et al., 2010;Rull, 2008Rull, , 2011aRull, , 2011b. Different hypotheses were suggested for the origin and maintenance of biodiversity in this region, such as the isolation of South America, the Andean uplift, the formation of the Isthmus of Panama land bridge, and the Quaternary climatic fluctuations (Batalha-Filho et al., 2012;Fouquet, Loebmann, et al., 2012;Fouquet, Recoder, et al., 2012;Fouquet, Recoder, et al., 2012;Paz et al., 2019). The original idea, considering the richness of tropical species as a result of a long-term process in stable environments, was abandoned with the refugia hypothesis, and most of the speciation was attributed to Quaternary events (Haffer, 1969(Haffer, , 2001. More recently, growing evidence of pre-Quaternary differentiation has accumulated, attributing tectonic, eustatic, and orogenic events (Geurgas et al., 2008;Ribas et al., 2009;Rull, 2008), or rivers as barriers to gene flow (Gascon et al., 2000;Passoni et al., 2008;Pellegrino et al., 2005).
The Amazonia and Atlantic Forest ecoregions in South America (Ab'Saber, 1977;Dinerstein et al., 2017) comprise the most diverse tropical forests in the world and are separated by the diagonal of open formations (Prado & Gibbs, 1993;Silva et al., 2004), which acts as a climatic barrier to species migration between these forested regions (Costa, 2003;Mori et al., 1981;Por, 1992). The "diagonal of open formations," also known as "the main South American disjunction" (Brieger, 1969), began to emerge during the Oligocene (Hoorn, Wesselingh, ter Steege, et al., 2010;Perret et al., 2013;Sobral-Souza et al., 2015). At the end of the Miocene (11-5 Mya), the increase in aridity was responsible for the rapid expansion of savanna vegetation and the separation of forests, remotely continuous, in two separate regions, the Amazonia to the West and the Atlantic Forest to the East, fully formed in the Pleistocene (2 Mya) (Arruda et al., 2018;Costa et al., 2018;Roig-Juñent et al., 2006;Sobral-Souza et al., 2015;Wesselingh & Salo, 2006). Currently, this dry corridor comprises the Chaco, the Pantanal, the Cerrado, and the Caatinga, Neotropical savannas, and seasonally dry forests (Ab'Saber, 1977;Sobral-Souza et al., 2015). Such separation means that both forests have few species or species groups in common (e.g., Lithobates palmipes, Rhinella margaritifera group; Pristimantis conspicillatus group), and several clades endemic to each region (Fouquet, Recoder, et al., 2012).
However, the two forested ecoregions have already been connected during the climatic fluctuations of the Neogene and the Quaternary period, which results in conflicting biogeographic relationships between the Eastern/Western Amazonia and the North and South of the Atlantic Forest. In addition, in relation to the animal composition, the Eastern Amazonia is more similar to the Northern Atlantic Forest and the Western Amazonia is more similar to the Southern Atlantic Forest (Cheng et al., 2013;Costa et al., 2018;Fiaschi & Pirani, 2009;Perret et al., 2006;Santos et al., 2009).
With the Andean uplift, the neotropical landscape had multiple changes, as in this period (Paleogene-Neogene) there was a drastic change in the climate (Insel et al., 2010) and the Amazon basin and the Pebas system were formed (a large wetland of shallow lakes and swamps developed in the Western Amazonia), creating new habitats that influenced the diversification of different groups, mainly in the Amazonia ecoregion (Antonelli et al., 2009;Hoorn, 1993;Hoorn, Wesselingh, Hovikoski, et al., 2010;Hoorn, Wesselingh, ter Steege, et al., 2010). The Atlantic Forest ecoregion, located in the eastern of South America, had significant changes with the global climate transition during the Cenozoic (Carnaval & Moritz, 2008). These climatic changes in the Atlantic Forest influenced the diversification of groups in different ecoregions (Antonelli et al., 2010;Graham et al., 2010;Hughes et al., 2013) and promoted the evolution of recent lineages of diverse groups of animals and plants within the forest ecoregion (Carnaval et al., 2009;Fitzpatrick et al., 2009;Mata et al., 2009;Porto et al., 2013;Thomé et al., 2014).
Despite the limited dispersion of amphibians, some lineages are distributed in both Amazonia and Atlantic Forest regions, as the treefrog tribe Sphaenorhynchini. Within the tribe, the single species K E Y W O R D S dispersal, Gabohyla, hatchet-faced tree frog, lime Tree Frogs, phylogeny, short-snouted green tree frogs, Sphaenorhynchus, zoogeography
Given the distribution of this clade restricted to the two largest forests ecoregions in South America, combined with the known evolutionary history between these two domains, here we report the results of a study that inferred a dated phylogenetic relationship through a Bayesian analysis among species of the Sphaenorhynchini tribe using mitochondrial and nuclear markers throughout Atlantic Forest and the Amazonia in South America. We aimed to evaluate historical biogeographical scenarios that can explain the current Sphaenorhynchini distribution. Once the Atlantic Forest harbors more species within this tribe, we hypothesized that Sphaenorhynchini originated in this ecoregion and that during the Miocene, when there was a connection between both domains, some species have dispersed to Amazonia, and subsequently vicariant into numerous widely disparate populations.

| Sample collection, alignment editing, and generation
We used 12 species of the genus Sphaenorhynchus and one of Gabohyla derived from previous phylogenetic studies ( Araujo-Vieira et al., 2019, 2020Faivovich et al., 2005;Wiens et al., 2006) S. prasinus, and S. surdus. All sequences are available at Genbank (https://www.ncbi.nlm.nih.gov/genba nk/; Appendix S1). There are no sequences available for Atlantic Forest species Sphaenorhynchus bromelicola and S. palustris, and therefore were not included in our analyses. We obtained sequences from 41 specimens from the Sphenorhynchini tribe in addition to Scinax fuscovarius which was used as an outgroup (Hime et al., 2021; see Appendix S1).
For phylogenetic analysis, we used four molecular markers (see Appendix S1), three mitochondrial genes (12S, 16S, and Cytochrome b-Cytb), and two nuclear genes (Recombination Activating 1-Rag1 and Tyrosinase-Tyr). This led to an alignment of 687 base pairs (bp) for the 12S gene, 1,053 bp for the 16S gene, 299 bp for the Cytb gene, 370 bp for Rag1, and 432 bp for the Tyr gene. The sequences were aligned with the ClustalW algorithm (Sievers et al., 2011) and visually verified in the Geneious v9.1.2.

| Phylogenetic analysis
To estimate the best substitution model for each gene segment, we used the Bayesian Information Criterion (BIC, Sullivan & Joyce, 2005) implemented in the jModelTest 2.1.4 program (Darriba et al., 2012).
The most suitable models were GTR+I+G for 12S, GTR+I+G for 16S, HKY+I+G for Cytb, K80+G for Rag1, and K80+G for Tyr. To infer the timing of speciation events within the tribe Sphaenorhynchini, we built a species tree in *BEAST using the three mitochondrial and two nuclear genes in BEAST v2.6.3 (Bouckaert et al., 2019). Due to the lack of fossil calibrations for this group, we used the 16S mutation  (Crawford, 2003). We ran 300 million generations, sampling every 30,000 steps using a tree from the Yule Process prior. We visually evaluated the convergence of the MCMC executions and the effective sample sizes (ESS values ≥200) using the TRACER 1.7 program (Rambaut et al., 2018). The first 10% of sampled genealogies were discarded as burn-in, and the most credible clade was inferred with TreeAnnotator v2.6.3 (Bouckaert et al., 2019).

| Biogeographical analysis
The geographical distribution over time of the Sphaenorynchini tribe in South America was estimated with the 'BioGeoBEARS' package (Matzke, 2013(Matzke, , 2014 in the R environment (R Core Team, 2020). The  Ronquist, 1997), and BayArea (Bayesian Inference from Historical Biogeography for Discrete Areas; Landis et al., 2013). These three methods were originally developed in different structures (Probability for DEC, Parsimony for DIVA, and Bayesian for BayAREA), but they are all represented as probability models in "BioGeoBEARS" to allow direct comparison. The latter two models are therefore not identical to their original formulation and are referred to as DIVALIKE and BAYAREALIKE within "BioGeoBEARS" (Matzke, 2013). Collectively, these models allow for a wide range of processes, including speciation within the area, vicariance, range expansion (dispersion to a new area), and range contraction (extinction in an area). We also tested models with and without founder event speciation, which is incorporated into parameter j. From the model with the lowest Akaike information criterion (AIC), we estimate the probabilities of the ancestral area along the phylogeny (Burnham & Anderson, 2003).
In DEC, the geographic range is allowed to change across a phylogeny through several types of events. Along the branches of a phylogenetic tree (anagenetic evolution), the events allowed are "dispersal" (range expansion by adding an area) and "extinction" (range reduction through extirpation in an area), and these are treated as continuous-time Markov processes (Matzke, 2014). In general, the three "+ j" models significantly improved the fit of the model to the corresponding model without the inclusion of "+ j." For Sphaenorynchini, under DEC + j, the j parameter is always positive, and the d and e parameters are inferred to be closer to zero. This is an indication that the "D" and "E" processes of the DEC model are unnecessary for explaining the biogeography of Sphaenorynchini.
Instead, the data are explained with a much higher probability by a series of founder events. We are aware of the critique of DEC/DEC + j statistical comparisons put forward by Ree and Sanmartín (2018), but we decided to maintain this parameter based on the replies indicating the statistically invalid presented by Klaus and Matzke (2020) and Matzke (2021) about DEC/DEC + j comparisons.

| Phylogeny and divergence times
In the Bayesian analysis, 58% of the nodes were strongly supported ( Figure 2). Most speciation events that reproduce the current diversity occurred between 19 and 6 million years ago, and the greatest   (Figure 2). The initial division of existing species in this group was estimated during the Miocene period (9.52 Mya; 95% HDP: 7.79-11.29 Mya). In this same period, there was a divergence between the two species of the S. planicola group (S. mirim and S. planicola), dated 6.4 Mya (95% HDP: 1.96-10.32 Mya).

| Ancestral area estimates
The DEC model, through a founding event (+ j), was the most adequate to the data (AIC = 54.70; Table 1). Our results indicate that the first Sphaenorhynchini diversification event occurred in the North Atlantic Forest, about 19 million years ago, at the beginning of the Miocene (also supported by models without the "+J" parameter, Figure 3). In general, the three "+J" models improved modelfit significantly for the corresponding model without the inclusion of "+J." Similar scenarios were also obtained by the DIVALIKE+J model, which was the second-best model inferred from our data by BioGeoBEARS (AIC = 56.01; Table 1   The differences found among our study and Araújo-Vieira et al. (2019, 2020) in relation to the species tree may be due to the different approaches used in the studies, where we used Bayesian phylogenetic analysis and the last used the parsimony method. Maximum Parsimony (MP) is a method that tries to minimize the number of mutations because it considers that one mutation is more likely than two. It is a discrete method and does not use probabilistic evolution models (Garcia, 2007). The major problem with this method is that it fails to take into account many sequences evolution factors (e.g., reversals, convergence, and homoplasy). Thus, the deeper the divergence times the more likely these methods will lead to erroneous or poorly supported groupings. Bayesian Inference (BI) is based on a posteriori probability, using an a priori probability and generating a phylogenetic tree according to the data. Supposedly infers trees with high support for clades, provides a distribution of trees that allows the choice of hypotheses (trees) with greater posterior probability (Lewis et al., 2005;Mar et al., 2005). One of the most appealing aspects of Bayesian phylogenetic inference is its presentation and comparison of multiple optimal hypotheses. While a MP attempts to produce the shortest topologies, BI produces a range of solutions, each with a corresponding overall posterior probability as well as comparable node support values for alternative topologies within each tree hypothesis (Li, 1996;Mau et al., 1999). Some studies have also suggested that BI trees have a higher resolution than MP (Spencer & Wilberg, 2013 (Linder, 2008;Perret et al., 2013;Rull, 2011a).
The MRCA of the Sphaenorhynchini species emerged in the north of Atlantic Forest and migrated to the Amazonia in different dispersion events that occurred during the connections between these ecoregions. After the Cretaceous-Paleogene extinction event, paleoclimatic and palynological analyses (e.g., Costa, 2003;Ledru, 1993;Micheels et al., 2007;Ortiz-Jaureguizar & Cladera, 2006;Sobral-Souza et al., 2015) indicate that the climate of South America was humid and hot during much of its range in the Paleogene, due to the PETM (Paleocene-Eocene Thermal Maximum). This climate would have promoted forest development across the continent, allowing the Amazonia and Atlantic Forests to be connected (Costa, 2003;de Oliveira et al., 1999;Patton et al., 1997;Wang et al., 2004;Willis, 1992) through different biogeographic routes (see Por, 1992). However, during the Eocene-Oligocene (~34 Mya) the climate began to undergo sudden changes due to the isolation of the Antarctic (Carter et al., 2017;Goldner et al., 2014;Kvasov & Verbitsky, 1981), causing global cooling. The climatic fluctuations continued through the Oligocene and Miocene (Graham et al., 2010;Jaramillo et al., 2010;Zachos et al., 2001) changing the composition of vegetation worldwide (Meseguer et al., 2015) and probably caused the contraction and the rupture of previously continuous tropical forest areas , The idea of forest corridors connecting the Eastern Amazonia and the Northeast Atlantic Forest (Ledo & Colli, 2017;Melo Santos et al., 2007;Rizzini, 1963) are corroborated with the biogeographic standards that we found for species of the tribe Sphaenorhynchini.
Although the "Dry Diagonal of Open Formations" has limited migration between the Amazonia and Atlantic Forest, which are important in the diversification of amphibians and reptiles (Castroviejo-Fisher et al., 2014;Fouquet, Loebmann, et al., 2012;Fouquet, Recoder, et al., 2012;Prates et al., 2016;, gallery forests and more humid portions, usually in high altitude areas, maintained some connectivity between forest ecoregions (Costa, 2003;Fine & Lohmann, 2018;Ledo & Colli, 2017;Sobral-Souza et al., 2015). For example, fossils, paleopalinological data, and speleothems from the Caatinga ecoregion in Northeastern Brazil indicate that in the past xeric vegetation was replaced by species of tropical forest trees, due to higher levels of precipitation (Auler & Smart, 2001;Auler et al., 2004;Cartelle & Hartwig, 1996;Czaplewski & Cartelle, 1998;de Oliveira et al., 1999;Wang et al., 2004). Furthermore, the existence of the Caatinga enclaves' moist forests, which are forest entrances within the semiarid vegetation of the Caatinga ecoregion, forming islands of humid forest (Andrade-Lima, 1982) containing a mixture of species with Amazonia and Atlantic affinities supports the hypothesis of forest corridors (Mângia et al., 2018). Other potential factors also influenced the diversification patterns, such as geological history (tectonic movements and mountain orogenesis), which had a profound consequence for the origin and evolution of Neotropical biodiversity by increasing and breaking of biogeographic barriers (Antonelli et al., 2009;Moritz et al., 2000). For example, along the Neogene  (Hoorn, Wesselingh, ter Steege, et al., 2010).
Thus, it may have restricted S. dorisae in the Western Amazonia, between the Madeira and Negro Rivers while S. lacteus was widely distributed, associated with the entire Amazonia basin, also occurring in transition zones between the Amazonia and the Cerrado (see Silva et al., 2020) and in gallery forests, between Cerrado and Caatinga, in Northeastern Brazil (see Benício et al., 2011). The wide distribution of this species is a curious fact and may be due to its ancient diversification, but only with a phylogeographic study covering the entire distribution of this species, it will be possible to understand which historical events influenced the current geographical distribution.
Following the same pattern, in the Atlantic Forest is evident that diversity is highly structured along a North-South gradient, and that rivers probably played an important role in this divergence (Behling, 1997;Jackson, 1978;Ledru et al., 2005;Oliveira-Filho & Fontes, 2000;Pellegrino et al., 2005).  Amaro et al., 2012;Batalha-Filho et al., 2012;Carnaval et al., 2009Carnaval et al., , 2014Costa, 2003;D'Horta et al., 2011;Grazziotin et al., 2006;Pellegrino et al., 2005;Pirani et al., 2020;Resende et al., 2010;Thomé et al., 2010;Valdez & D'Elía, 2013). It is possible to observe some of these barriers in the distribution of Sphaenorhynchini species, such as, for example, the Paraíba do Sul River valley in Rio de Janeiro and Minas Gerais states and the Rio Doce River in Espiríto Santo and Minas Gerais states, which together, is the current limit from the distributions of S. canga (see Araujo-Vieira et al., 2015), S. prasinus (see da Silva et al., 2013), S. pauloalvini (see Freitas et al., 2009), and S. mirim (see Caramaschi et al., 2009. The Tietê River region in São Paulo state, is the current limit of the S. caramaschi distribution (see Melo et al., 2018), S. surdus (see Toledo et al., 2007) and may have played an important role in the divergence of these species from the ancestor of the other species of Sphaenorhynchini.

Despite the role of rivers as barriers in Amazon and in Atlantic
Forest, recent studies have associated that the climatic fluctuations of the Pleistocene induced the fragmentation of the forest formations (Cabanne et al., 2008;Carnaval et al., 2014;Thomé et al., 2010), isolating limited-dispersal organisms. The remaining forest fragments would be isolated and, in these forest refuges, new species would emerge from the widely distributed ancestral species (Carnaval & Moritz, 2008;Martins, 2011). The Pleistocene refuges (Carnaval et al., 2009;Haffer, 1969Haffer, , 1997Vanzolini & Williams, 1981) have been used and reviewed as a scenario that explains what caused the increase in the diversity and richness rate of these environments (Bush & Oliveira, 2006;Connor, 1986;Garzón-Orduña et al., 2015). botocudo, and G. pauloalvini (see Caramaschi et al., 2009;de Freitas et al., 2009) and S. caramaschii occurring in the São Paulo refugium (see Melo et al., 2018). However, the divergence times between species are older than the LGM. Thus, the Miocene climate changes may have played a central role in the simultaneous origin of these taxa, showing that some of these forest fragments remained relatively stable for a much longer period than that proposed by Carnaval and Moritz (2008), thus serving as a successive refuge in the climatic cycle. Phylogeographic studies, covering the area of occurrence of all species of the Sphaenorhynchini tribe, with extensive sampling, will clarify and allow a better understanding of these patterns.

ACK N OWLED G M ENTS
We would like to thank two anonymous reviewers and the editor Chris Foote for helping to improve the final version of our paper. This study was funded in part by the Coordenação de Aperfeiçoamento

DATA AVA I L A B I L I T Y S TAT E M E N T
The alignments used in this study is available at Github Digital  (2)